Method for viewing tumor tissue located within a body cavity

ABSTRACT

Methods are provided for in vivo detection of tumor tissue associated with a disease state in a subject, such as tumor tissue located in body opening. In the invention method, the subject is administered a biologically compatible fluorescing targeting construct, the construct is allowed to bind to and/or be taken up by tumor tissue present in the subject, a body part of the subject suspected of containing the tumor tissue is irradiated with UV light while extraneous light to the body part is substantially eliminated, and fluorescence emanating from the fluorescing targeting construct bound to and/or taken up by the tumor tissue is directly viewed by the observer with or without the aid of an endoscope, so as to determine the location and size of the tumor tissue. The invention methods offer the advantage that diseased or abnormal tissue can be directly viewed at interior body sites with or without the aid of an endoscopic device, and without the use of additional imaging equipment, for example, through a surgical opening to facilitate a procedure of biopsy or surgical excision.

RELATED APPLICATIONS

This application is a Continuation-In-Part application of copending U.S.patent application Ser. No. 09/173,190, filed Oct. 15, 1998, entitled“Method For Viewing Diseased Tissue Located Within A Body Cavity,” whichis incorporated herein by reference in its entirety.

FIELD OF THE INTENTION

The present invention relates to methods for viewing the state of a bodycavity or an internal organ of a mammalian body. More particularly, theinvention relates to a method for detecting tumor tissue at an interiorbody site using a fluorescent tumor-avid ligand excited by UV light.

BACKGROUND OF THE INVENTION

Many solid and liquid substances naturally emit fluorescent radiationwhen irradiated with ultraviolet light. However, the radiation may fallwithin wide wavelength bands of low intensity. In the case of manynatural objects, observations are partially obscured by naturalfluorescence emanating simultaneously from many different compoundspresent in the sample under examination. In imaging devices such asmicroscopes, therefore, it is known to employ a filter for a selected UVwavelength band to screen out undesired fluorescence emanating from theobject under observation.

In medical applications, a similar difficulty arises because both tumorsand healthy tissue fluoresce naturally, albeit at different wavelengths.Consequently, when UV-activated fluorescence is used to detect tumorsagainst a background of healthy tissue, identification of tumors isdifficult. However, unlike most other cells of the body, tumor cells maypossess a natural ability to concentrate and retain hematoporphyrinderivative dyes. Based upon this discovery, a technique was developedwherein a hematoporphyrin derivative fluorescent dye is administered andallowed to concentrate in a tumor to be examined to increase thefluorescence from the tumor as compared with that of healthy backgroundtissue. Hematoporphyrin dyes fluoresce within a fluorescence spectrumbetween 610 and 700 nm, a spectrum easy to detect. However, the naturalfluorescence from healthy in cells is still much more intense than thatfrom the dyes, and has a broader fluorescence spectrum. Thus, the use offluorescent dyes in diagnosis of tumors has not been wholly successful.

In endoscopic systems, it is also known to irradiate an internal organwith visible radiation to obtain a visible image and then to apply tothe internal organ a fluorescent dye that concentrates in tumors over aperiod of time. The dye is allowed to concentrate, and then the internalorgan is irradiated with excitation radiation for the dye to obtain asecond fluorescent image. A body part having abnormal or diseasedtissue, such as a cancer, may be identified by comparing an imageproduced by visible radiation of the internal organ with the imageproduced by fluorescence. To aid in visualizing the images received,endoscopic systems commonly utilize a television camera attached to afiber optic scope having an optical guide fiber for guiding a beam froman external radiation source to the internal organ, and another opticalguide fiber for transmitting a fluorescent image of the affected area toa television monitor for viewing. These two approaches are combined in amethod of the type disclosed in U.S. Pat. No. 4,821,117, wherein afluorescent dye is applied to an object to be inspected, allowed toconcentrate in the tumor, and the affected site is then alternatelyirradiated with visible light and with radiation at the excitationwavelength of the fluorophore. Images of the object obtainedindependently by visible and fluorescent light using a TV camera arestored in memory, and are simultaneously displayed in a televisionmonitor to visually distinguish the affected area of the body part fromthe healthy background tissue.

In another type of procedure, such as is described in U.S. Pat. No.4,786,813, a beam-splitting system splits the fluorescence radiationpassing though the optical system into at least three parts, each ofwhich forms a respective image of the object corresponding to each ofthe wavelength regions received. A detector produces a cumulativeweighted signal for each image point corresponding to a single point onthe object. From the weighted signal values of the various points on theobject, an image of the object having improved contrast is produced.This technique is used to aid in distinguishing the fluorescence fromthe affected tissue from that produced by normal tissue.

A still more complex method of visualizing images from an endoscopicdevice uses television scanning apparatus. For example, U.S. Pat. No.4,719,508 discloses a method utilizing an endoscopic photographingapparatus wherein the endoscope includes an image sensor forsuccessively generating image signals fed to a first frame memory forstoring the image signals and a second frame memory for interlacing andstoring image signals read successively from the first frame memory. Thestored, interlaced image signals are delivered to a TV monitor fordisplay to aid in visualizing the affected body part.

These prior art endoscopic systems, which rely on photographic imagingof the area of interest (i.e., via a TV monitor), while effective, havehistorically relied on increasingly complex and expensive equipment andsubstitute indirect viewing for direct viewing of the affected bodypart.

Certain of the fluorescent dyes that concentrate in tumors due tonatural bodily processes can be excited at wavelengths corresponding tothose produced by lasers to accomplish diagnostic and therapeuticpurposes. Consequently, lasers have also been used in proceduresutilizing endoscopic systems in conjunction with fluorescent dyes toimage and treat tumors. In one embodiment of this general method, a dyeis used that absorbs laser light at two different wavelengths and/orlaser powers, one that excites fluorescence without generating damagingheat in the tissue, and one that generates sufficient heat in the dye todestroy surrounding tissue. U.S. Pat. No. 4,768,513, for example,discloses a procedure in which a dye is applied to a body part suspectedof containing a tumor, usually by local injection. The dye is allowed toconcentrate in tumors and clear from healthy tissue over a period ofdays, and then the body part is irradiated with alternate pulses of twolight sources: a white light of a known intensity and afluorescence-exciting laser light. To compensate for variations inintensity of the fluorescence resulting from variations in the angle ofincident light, and the like, visualization of the tumor iscomputer-enhanced by calculating the intensity of the fluorescence withrespect to the known intensity of the white light. Ablation of a tumordetected using this method is accomplished by switching the laser to theheat-generating wavelength so as to destroy the cancerous tissue intowhich the fluorophore has collected.

While effective for diagnosing and treating tumor, such methods have twomajor drawbacks. Disease states other than tumor cannot be diagnosed,and laser visualization must be delayed for a period of two days or moreafter administration of the fluorescent dye to allow the dye to clearfrom normal tissue.

Monoclonal antibodies and other ligands specific for tumors have beendeveloped for use in diagnosis of tumors, both in tissue samples and invivo. In addition to such ligands, certain tumor-avid moieties aredisproportionately taken up (and optionally or metabolized by tumorcells). Two well-known tumor-avid compounds are deoxyglucose, whichplays a telling role in glycolysis in tumor cells, and somatostatin,which binds to and/or is taken up by somatostatin receptors in tumorcells, particularly in endocrine tumors.

In such studies, deoxyglucose is used as a radio-tagged moiety, such asfluorodeoxyglucose (¹⁸F-deoxyglucose), for detection of tumors ofvarious types. It is believed that tumor cells experience such amismatch between glucose consumption and glucose delivery that anaerobicglycolysis must be relied upon, thereby elevating the concentration ofthe radioactive tag in tumor tissue. It is also a possibility that theelevated concentration of deoxyglucose in malignant tumors may be causedby the presence of isoenzymes of hexokinase with abnormal affinities fornative glucose or its analogs (A. Gjedde, Chapter 6: “GlucoseMetabolism,” Principles of Nuclear Medicine, 2nd Ed., W. B. SaundersCompany, Philadelphia, Pa., pages 54-69). Similarly, due to theconcentration of somatostatin in tumor tissue, radio-taggedsomatostatin, and fragments or analogs thereof, are used in the art fornon-invasive imaging of a variety of tumor types in a procedure known assomatostatin receptor scintigraphy (SRS).

Although these techniques have met with considerable success indetermining the presence of tumor tissue, scintigraphic techniques aredifficult to apply during a surgical procedure because of the equipmentnecessary for viewing the image provided by the radioisotope. Yet it isexactly at the time that the surgeon has made the incision or enteredthe body cavity that it would be most useful to “see” the outlines ofthe diseased tissue.

Thus, there is a need in the art for new and better methods that can beused to directly visualize a broad range of putative disease sites usingfluorophores that are excited by UV light with or without the use ofendoscopic instruments. Where visualization is by means of endoscopicdevices, direct visualization (as opposed to creation of photographicimages) offers the additional advantage that the equipment required iscomparatively simple to use and is less expensive than the equipmentrequired to create photographic displays from such images. In addition,there is a need in the art for a method of identifying diseased orabnormal tissue during surgical procedures so that immediate resectionor biopsy of the identified tissue can be performed while the surgeon“sees” the outlines of the diseased or abnormal tissue.

SUMMARY OF THE INVENTION

The present invention provides method(s) for in vivo identification oftissue associated with a disease state in a subject in need thereof. Theinvention method(s) comprise administering to the subject adiagnostically effective amount of a fluorescing targeting constructcomprising a tumor-avid compound so as to allow the fluorescingtargeting construct to bind to and/or be taken up by the target tumortissue, irradiating a body part of the subject suspected of containingthe target tumor tissue with UV light having at least one wavelength inthe excitation spectrum of the targeting construct under conditions thatsubstantially eliminate extraneous light to the body part, and directlyviewing fluorescence emanating from the fluorescing targeting constructbound to the target tumor tissue so as to determine the location and/orsurface area of the target tumor tissue in the body part. Tumor-avidcompounds that are preferentially “taken up” by tumor cells and can beused as the ligand moiety in the invention targeting constructsgenerally enter the cells through surface or nuclear receptors, such ashormone receptors, or through pores, hydrophilic “windows” in the celllipid bilayer, and the like.

If the putative body part is a body opening of the subject or asurgically produced interior site, an endoscopic device is optionallyused to direct the UV light to the body part and to receive fluorescencefrom the fluorescing targeting construct for direct viewing. Thus, anendoscopic device can aid in detection of tumor tissue associated with adisease state by direct viewing of fluorescence emanating from thetumor-avid ligand attached to or taken up by the target tumor tissue.

In a presently preferred embodiment of the invention method, thefluorescing targeting construct comprises a biologically compatibleUV-sensitive fluorescing moiety and a tumor-avid ligand moiety thatpreponderantly binds to and/or is taken up by tumor tissue, such asdeoxyglucose, somatostatin, a somatostatin receptor-binding peptide,methionine, and the like. Additional tumor-avid ligands are hormones andother compounds which bind to and/or are taken up preferentially bytumor cells, for example by receptors, such as nuclear receptors,expressed by tumor cells. The tumor can be any type for which a specifictumor-avid ligand is known or can be pre-determined using screeningprocedures described herein.

The invention method is particularly suited to in vivo detection oftumor tissue located at an interior site in the subject, such as withina natural body cavity or a surgically created opening, without the needfor an endoscopic device. For example, the tumor tissue can becontemporaneously viewed through a surgical opening to facilitate aprocedure of biopsy or surgical excision. As the precise location and/orsurface area of the tumor tissue are readily determined by the inventiondiagnostic procedure, the invention method is a valuable guide to thesurgeon, who needs to “see” the exact outlines, size, etc. of the massto be resected as the surgery proceeds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for in vivo identification oftissue associated with a disease state in a subject in need thereofusing a fluorophore sensitive to UV light. The invention methodcomprises administering to the subject a diagnostically effective amountof a fluorescing targeting construct comprising a tumor-avid moiety soas to allow the fluorescing targeting construct to bind to and/or betaken up by the target tissue, irradiating a body part of the subjectsuspected of containing the target tissue with UV light having at leastone wavelength in the excitation spectrum of the targeting constructunder conditions that substantially eliminate extraneous light to thebody part, and directly viewing fluorescence emanating from thefluorescing targeting construct bound to or taken up by the targetingtissue so as to determine the location and/or surface area of the targettissue in the body part.

If the putative disease site is a natural body cavity or surgicallyproduced interior site, an endoscopic device can be optionally used todeliver the excitation UV light to the site, to receive fluorescenceemanating from the site, and to aid in formation of a fluorescent imageof the fluorescence from the diseased tissue. For example, a lens in theendoscopic device can be used to focus the detected fluorescence as anaid in formation of the image. Alternatively, the UV light may bedirected into a body cavity or surgical opening by any convenient meansand the fluorescent image so produced can be directly visualized by theobserver without aid from an endoscope.

In operating rooms it is possible to have an overhead UV light of thedesired optical emitting spectrum. Such a light can be utilized in thepractice of the invention merely by turning out the other lights in theoperating room (to eliminate extraneous light that would be reflectedfrom the body part under investigation) and shining the UV light intothe body cavity or surgically created opening so that the fluorescentimage received directly by the eye of the observer (e.g., the surgeon)is predominantly the fluorescent image emanating from the fluorophore(s)in the field of vision on the selected body part. In any event, to aidin accomplishing the goal of direct visualization by the observer,extraneous light to the body part of interest is substantiallyeliminated so that light reflected from the body part, other than thatfrom the UV-sensitive fluorescent moieties, is minimized or eliminated.With or without aid from any type of endoscopic device, the fluorescentimage produced by the invention method is such that it can be viewedwithout aid of a camera, TV monitor, or other such additional equipment.For example, the diseased or abnormal tissue can be contemporaneouslyviewed through a surgical opening to facilitate a procedure of biopsy orsurgical excision. As the location and/or surface area of the diseasedtissue are readily determined by the invention diagnostic procedure, theinvention method is a valuable guide to the surgeon, who needs to knowthe exact outlines, size, etc. of the mass to be resected as the surgeryproceeds.

In one embodiment of the invention method, a single type of fluorescentmoiety is relied upon for forming the fluorescence emanating from theirradiated body part. Since certain types of healthy tissue fluorescenaturally, in such a case it is important to select a fluorescent moietyfor the targeting construct that has a predominate excitation wavelengthnot shared by the healthy tissue found in the body part to beilluminated by the UV light. In this embodiment of the invention, theirradiating UV light from the light source may be monochromatic orpolychromatic with at least one irradiating wavelength matched to thepredominant excitation wavelength of the fluorescent targetingconstruct. By this means, the amount of fluorescence emanating fromhealthy “background” tissue in the field of vision is minimized.

In alternative embodiments, the invention method may additionallycomprise the step of administering to the subject one or moresupplemental fluorescing targeting constructs (e.g., antibodies, orbiologically active fragments thereof, having attached fluorophores)that bind to the initial fluorescent targeting construct and/or to eachother to enhance the fluorescence emanating from the target tissue. Forinstance, a fluorescently tagged anti-fluorophore antibody may beadministered to bind to any previously administered fluorescently-taggedantibody or tumor-avid molecule. The purpose of the supplementalfluorescing targeting construct is to increase the intensity offluorescence from the targeting ligand of the first administeredtargeting construct and thereby to aid in detection of diseased orabnormal tissue in the body part.

It is generally good practice to allow the targeting construct to bindto and/or be taken up by any targeting tissue that may be present at thesite under investigation and then, before administration of thesupplemental fluorescing targeting construct(s), to substantially remove(e.g., wash) from the body part any unbound targeting construct tomaximize the opportunity for fluorescence from the supplementalfluorescing targeting constructs(s) to aid in detecting the presence ofany target tissue present in the body part. Usually, the supplementalfluorescing targeting constructs are successively administered to buildup the fluorescent signal from the target tissue. For example, if thefluorescent targeting construct comprises a humanized IgG monoclonalantibody specific for a breast cancer antigen, the next-administeredfluorescing targeting construct may comprise an anti-fluorophoreantibody, such as anti-fluorescein, and the third-administeredfluorescing targeting construct may comprise an anti-idiotype antibody.Those of skill in the art will be able to devise combinations ofsuccessively administered fluorescing targeting constructs, each ofwhich specifically binds to the targeting construct or to one or more ofthe earlier administered supplemental fluorescing targeting constructs.It is presently preferred that all of the fluorescing targetingconstructs used to identify the target tissue comprise fluorophores thatfluoresce within the same wavelength band or at the same wave length asdoes the initially administered targeting construct (e.g. theUV-sensitive fluorescing moiety in the initially administered targetingconstruct) to minimize the number of different UV sources that need tobe employed to excite simultaneous fluorescence from all of thedifferent targeting constructs used in practice of the invention method.

In yet another embodiment, the invention method further comprises thestep of administering to the subject at least one supplementalfluorescent targeting construct (e.g., comprising an antibody, or abiologically active fragment thereof having an attached fluorophore)that specifically binds to or is taken up by normal tissue or constructsin the body part, wherein fluorescence from the supplemental fluorescenttargeting construct(s) in response to the irradiating UV light is adifferent color (i.e., has a different wavelength) than that from theflorescent targeting construct that is selected to bind to or be takenup by the target tissue. The difference in the colors of thefluorescence emanating from fluorophores in targeting constructstargeted to normal and to diseased or abnormal target tissue aids theobserver in determining the location and size of the target tissue. Thisembodiment of the invention provides the advantage that any naturalfluorescence emanating from normal tissue is obscured by thefluorescence emanating from fluorophore(s) in supplemental targetingconstructs targeted to the normal tissue in the body part. The greaterthe difference in color between the fluorescence emanating from normaland target tissue, the easier it is for the observer to visualize theoutlines and size of the target tissue. For instance, targeting afluorescing targeting construct comprising a fluorophore producing redlight to the target tissue (i.e., abnormal tissue) and a fluorophoreproducing green light to healthy tissue aids the observer indistinguishing the target tissue from the normal tissue.

The spectrum of UV light used in the practice of the invention method isselected to contain at least one wavelength that corresponds to thepredominate excitation wavelength of the targeting construct, or of abiologically compatible UV-sensitive fluorescing moiety contained withinthe targeting construct. Generally the UV light used in practice of theinvention method comprises at least one wavelength of light in the UVwavelength range, for example in the range from about 4 nm to about 400nm, and preferably in the range from about 340 nm to about 400 nm. TheUV light may be monochromatic or polychromatic.

However, when a combination of targeting ligands that fluoresce atdifferent wavelengths is used in practice of the invention, the spectrumof the UV light must be broad enough to provide at least one excitationwavelength for each of the fluorophores used. For example, it isparticularly important when fluorophores of different colors areselected to distinguish normal from diseased tissue, that the excitationspectrum of the UV light(s) include excitation wavelengths for thefluorophores targeted to normal and target tissue.

The UV-sensitive fluorescing moiety of the targeting construct or of thesupplemental fluorescing targeting ligand(s) can be any chemical orprotein moiety that is biologically compatible (e.g., suitable for invivo administration) and which fluoresces in response to light having awavelength in the UV range selected. Since the targeting ligand isadministered to living tissue, biological compatibility includes thelack of substantial toxic effect to the individual in general, and tothe target tissue, in particular. Non limiting examples of UVsensitive-fluorophores that can be used in the practice of the inventioninclude quinine, fluorescein, tetracycline, Allura Red AC dye (alsoknown as D&C Red #40, having a chemical formula: disodium6-hydroxy-5-(6-methoxy-4-sulphonatom-tolylazolnaphthalene-2-sulphonate), phloxine B (also known as Acid red92 or D&C Red 28, having a chemical formula:spiro[isobenzofuran-1(3H),9′[9H]xanthen]-3-one,2′,4′5′,7′-tetrabromo-4,5,6,7-tetrachloro-3′,6′-dihydroxy-disodiumsalt), and the like, and combinations of two or more thereof.

Since the fluorescence properties of biologically compatiblefluorophores are well known, or can be readily determined by those ofskill in the art, the skilled practitioner can readily select a usefulfluorophore or useful combinations of fluorophores, and match thewavelength(s) of UV light to the fluorophore(s). Toxicity of additionaluseful fluorophores can be determined using animal studies as known inthe art.

Preferably, the targeting construct (e.g., the ligand moiety of theinvention targeting construct) is selected to bind to and/or be taken upspecifically by the target tissue of interest, for example to an antigenor other surface feature contained on or within a cell thatcharacterizes a disease or abnormal state in the target tissue. As inother diagnostic assays, it is desirable for the targeting construct tobind to or be taken up by the target tissue selectively or to an antigenassociated with the disease or abnormal state; however, targetingconstructs containing ligand moieties that also bind to or are taken upby healthy tissue or cell structures can be used in the practice of theinvention method so long as the concentration of the antigen in thetarget tissue or the affinity of the targeting construct for the targettissue is sufficiently greater than for healthy tissue in the field ofvision so that a fluorescent image representing the target tissue can beclearly visualized as distinct from any fluorescence coming from healthytissue or structures in the field of vision. For example, colon canceris often characterized by the presence of carcinoembryonic antigen(CEA), yet this antigen is also associated with certain tissues inhealthy individuals. However, the concentration of CEA in cancerouscolon tissue is often greater than is found in healthy tissue, so ananti-CEA antibody could be used as a ligand moiety in the practice ofthe invention. In another example, deoxyglucose is taken up and utilizedby healthy tissue to varying degress, yet its metabolism in healthytissues, except for certain known organs, such as the heart, issubstantially lower than in tumor. The known pattern of deoxyglucoseconsumption in the body can therefore be used to aid in determination ofthose areas wherein unexpectedly high uptake of deoxyglucose signals thepresence of tumor cells.

In one embodiment according to the present invention, the disease orabnormal state detected by the invention method can be any typecharacterized by the presence of a known target tissue for which aspecific binding ligand is known. For example, various heart conditionsare characterized by production of necrotic or ischemic tissue orproduction of artherosclerotic tissue for which specific binding ligandsare known. As another illustrative example, breast cancer ischaracterized by the production of cancerous tissue identified bymonoclonal antibodies to CA15-3, CA19-9, CEA, or HER2/neu. It iscontemplated that the target tissue may be characterized by cells thatproduce either a surface antigen for which a binding ligand is known, oran intracellular marker (i.e. antigen), since many targeting constructspenetrate the cell membrane. Representative disease states that can beidentified using the invention method include such various conditions asdifferent types of tumors, bacterial, fungal and viral infections, andthe like. As used herein “abnormal” tissue includes precancerousconditions, necrotic or ischemic tissue, and tissue associated withconnective tissue diseases, and auto-immune disorders, and the like.Examples of the types of target tissue suitable for examination usingthe invention method include cardiac, breast, ovarian, uterine, lung,endothelial, vascular, gastro-intestinal, colorectal, prostatic tissue,endocrine tissue, and the like, as well as combinations of any two ormore thereof.

Representative examples of antigens for some common malignancies and thebody locations in which they are commonly found are shown in Table Ibelow. Targeting ligands, such as antibodies, for these antigens areknown in the art.

TABLE I TUMORS WHERE ANTIGEN COMMONLY FOUND CEA (carcinoembryonicantigen) colon, breast, lung PSA (prostate specific antigen) prostatecancer CA-125 ovarian cancer CA 15-3 breast cancer CA 19-9 breast cancerHER2/neu breast cancer α-feto protein testicular cancer, hepatic cancerβ-HCG (human chorionic gonadotropin) testicular cancer, choriocarcinomaMUC-1 breast cancer Estrogen receptor breast cancer, uterine cancerProgesterone receptor breast cancer, uterine cancer EGFr (epidermalgrowth factor bladder cancer receptor)

In one embodiment of the invention method, the ligand moiety of thetargeting construct is a protein or polypeptide, such as an antibody, orbiologically active fragment thereof, preferably a monoclonal antibody.The supplemental fluorescing targeting construct(s) used in practice ofthe invention method may also be or comprise polyclonal or monoclonalantibodies tagged with a fluorophore. The term “antibody” as used inthis invention includes intact molecules as well as functional fragmentsthereof, such as Fab, F(ab′)₂, and Fv that are capable of binding theepitopic determinant. These functional antibody fragments retain someability to selectively bind with their respective antigen or receptorand are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow & Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference). Asused in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated in their entireties by reference. See also Nisonhoff etal., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119,1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422 AcademicPress, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and2.10.1-2.10.4. Other methods of cleaving antibodies, such as separationof heavy chains to form monovalent light-heavy chain fragments, furthercleavage of fragments, or other enzymatic, chemical, or genetictechniques may also be used, so long as the fragments bind to theantigen that is recognized by the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chainscan be lied by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Preferably,the Fv fragments comprise V_(H) and V_(L) chains connected by a peptidelinker. These single-chain antigen binding proteins (sFv) are preparedby constructing a structural gene comprising DNA sequences encoding theV_(H) and V_(L) domains connected by an oligonucleotide. The structuralgene is inserted into an expression vector, which is subsequentlyintroduced into a host cell such as E. coli. The recombinant host cellssynthesize a single polypeptide chain with a linker peptide bridging thetwo V domains. Methods for producing sFvs are described, for example, byWhitlow et al., Methods: a Companion to Methods in Enzyology, 2: 97,1991; Bird et al., Science 242:423-426, 1988; Pack et al.,Bio/Technology 11:1271-77, 1993; Sandhu, supra, and Ladner et al., U.S.Pat. No. 4,946,778, which is hereby incorporated by reference in itsentirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,Methods: a Companion to Methods in Enzymology, 2: 106, 1991.

Antibodies which bind to a tumor cell can be prepared using an intactpolypeptide or biologically functional fragment containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide used to immunize an animal (derived, for example, fromtranslated cDNA or chemical synthesis) can be conjugated to a carrierprotein, if desired. Commonly used carriers that are chemically coupledto the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin,bovine serum albumin (BSA), and tetanus toxoid, and the like. Thecoupled peptide is then used to immunize the animal (e.g., a mouse, arat, or a rabbit).

The preparation of such monoclonal antibodies is conventional. See, forexample, Kohler & Milstein, Nature 2:495, 1975; Coligan et al., sections2.5.1-2.6.7; and Harlow et al., in: Antibodies: a Laboratory Manual,page 726 (Cold Spring Harbor Pub., 1988), which are hereby incorporatedby reference. Briefly, monoclonal antibodies can be obtained byinjecting mice with a composition comprising an antigen, verifying thepresence of antibody production by removing a serum sample, removing thespleen to obtain B lymphocytes, fusing the B lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones that produce antibodies to the antigen, and isolating theantibodies from the hybridoma cultures. Monoclonal antibodies can beisolated and purified from hybridoma cultures by a variety ofwell-established techniques. Such isolation techniques include affinitychromatography with Protein-A Sepharose, size-exclusion chromatography,and ion-exchange chromatography. See, for example, Coligan et al.,sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al.,Purification of Immunoglobulin G (IgG), in: Methods in MolecularBiology, Vol. 10, pages 79-104 (Humana Press, 1992).

Antibodies of the present invention may also be derived from subhumanprimate antibodies. General techniques for raising therapeuticallyuseful antibodies in baboons can be found, for example, in Goldenberg etal., International Patent Publication WO 91/11465 (1991) and Losman etal., 1990, Int. J. Cancer 4:310, which are hereby incorporated byreference. Alternatively, a therapeutically useful antibody may bederived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementaritydetermining regions from heavy and light variable chains of the mouseimmunoglobulin into a human variable domain, and then substituting humanresidues in the framework regions of the murine counterparts. The use ofantibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833,1989, which is hereby incorporated in itsentirety by reference. Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522,1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992;Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol.150:2844, 1993, which are hereby incorporated by reference.

It is also possible to use anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

In a presently preferred embodiment of the invention method, the ligandmoiety in the fluorescent targeting construct used in practice of theinvention can be selected from among the many biologically compatibletumor-avid compounds that bind with specificity to receptors and/or arepreferentially taken up by tumor cells and can be used as the ligandmoiety in the invention targeting constructs. Tumor-avid compounds thatare preferentially “taken up” by tumor cells may enter the cells throughsurface or nuclear receptors (e.g., hormone receptors), pores,hydrophilic “windows” in the cell lipid bilayer, and the like.

Illustrative of this class of tumor-avid compounds are somatostatin,somatostatin receptor-binding peptides, deoxyglucose, methionine, andthe like. Particularly useful somatostatin receptor-binding peptides area long-acting, octapeptide analog of somatostatin, known as octreotide(D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-L-cysteinamidecyclic (2→7)-disulfide), lanreotide, an oral formulation of octreotide,P829, P587, and the like. Somatostatin-binding peptides are disclosed inU.S. Pat. No. 5,871,711, and methods for linking such peptidescovalently to a radioisotope through their carboxyl terminal amino acidunder reducing conditions are disclosed in U.S. Pat. No. 5,843,401,which are both incorporated herein by reference in their entireties. Oneof skill in the art can readily adapt such teachings for the preparationof UV sensitive somatostatin receptor-binding peptides by substitutingthe UV-sensitive fluorescing moieties of this invention in the place ofa radioisotope.

Somatostatin and somatostatin receptor-binding peptides are particularlyeffective for use as the tumor-avid ligand moiety in the targetingconstruct in the invention diagnostic procedures when the disease stateis a neuroendocrine or endocrine tumor. Examples of neuroendocrinetumors that can be diagnosed using the invention method include adenomas(GH-producing and TSH-producing), islet cell tumors, carcinoids,undifferentiated neuroendocrine carcinomas, small cell and non smallcell lung cancer, neuroendocrine and/or intermediate cell carcinomas,neuroendocrine tumors of ovary, cervix, endometrium, breast, kidney,larynx, paranasal sinuses, and salivary glands, meningiomas, welldifferentiated glia-derived tumors, pheochromocytomas, neuroblastomas,ganglioneuro(blasto)mas, paragangliomas, papillary, follicular andmedullary carcinomas in thyroid cells, Merkel cell carcinomas, andmelanomas, as well as granulomas and lymphomas. These tumor cells areknown to have somatostatin receptors and can be targeted usingsomatostatin or somatostatin receptor binding peptides as the tumor-avidligand in the invention fluorescent targeting construct.

Vasointestinal peptide (VIP), which is used in VIP receptor scintigraphy(I. Virgolini, Eur J. Clin. Invest. 27(10):793-800, 1997, is also usefulin the invention method for diagnosis of small primary adenocarcinomas,liver metastases and certain endocrine tumors of the gastrointestinaltract.

Another molecule illustrative of the tumor-avid ligands that arepreferentially taken up by tumors is deoxyglucose, which is known to bepreferentially taken up in a variety of different types of tumors.Illustrative of the types of tumors that can be detected usingdeoxyglucose as the tumor-avid ligand moiety in the fluorescenttargeting construct as disclosed herein include Preferred tumor targetsfor deoxyglucose include melanoma, colorectal and pancreatic tumors,lymphoma (both HD and NHL), head and neck tumors, myeloma, cancers ofovary, cancer, breast, and brain (high grade and pituitary adenomas),sarcomas (grade dependent), hepatoma, testicular cancer, thyroid (gradedependent) small cell lung cancer, bladder and uterine cancer, and thelike.

Yet other tumor-avid compounds that can be used as the targeting ligandin an invention fluorescing targeting construct are1-amino-cyclobutane-1-carboxylic acid and L-methionine. L-methionine isan essential amino acid that is necessary for protein synthesis. It isknown that malignant cells have altered methionine metabolism andrequire an external source of methionine.

Additional examples of biologically compatible tumor-avid compounds thatbind with specificity to tumor receptors and/or are preferentially takenup by tumor cells include mammalian hormones, particularly sex hormones,neurotransmitters, and compounds expressed by tumor cells to communicatewith each other that are preferentially taken up by tumor cells, such asnovel secreted protein constructs arising from chromosomal aberrations,such as transfers or inversions within the clone.

The term hormone is used herein to refer to compounds that are expressedwithin a mammal for action at a remote location and includes suchcompounds as sex hormones, cell growth hormones, cytokines, endocrinehormones, erythropoietin, and the like. As is known in the art, a numberof tumor types express receptors for hormones, for example, estrogen,progesterone, androgens, such as testosterone, and the like. Suchhormones are preferentially taken up by tumor cells, for example, viaspecific receptors. It is also known in the art that the particular typeof receptors expressed by a tumor cell may change over time with thesame cell or cell mass, for example, expressing estrogen receptors atone point in time and with the estrogen receptors being substantiallyreplaced with androgen receptors at another point in time.

Therefore, in another embodiment according to the present invention, theinvention diagnostic method comprises prescreening of target tumor cellsto determine which receptors are currently being expressed by the targetcells. In this embodiment, the invention diagnostic method comprisescontacting sample(s) of tumor cells obtained from a subject in vitrowith a plurality of detectably labeled tumor-avid compounds, anddetermining which of the tumor-avid compounds bind to or are taken up bythe sample cells. The invention diagnostic method further comprisesadministering to the subject a diagnostically effective amount of one ormore biologically compatible fluorescing targeting constructs, eachcomprising as ligand moiety at least one of the tumor-avid compoundsdetermined to bind to and/or be taken up by the tumor cells so as toallow the fluorescing targeting construct to bind to and/or be taken upselectively in vivo by tumor tissue, irradiating an in vivo body part ofthe subject suspected of containing the tumor tissue with UV lighthaving at least one wavelength in the excitation spectrum of thetargeting construct under conditions that substantially eliminateextraneous light to the in vivo body part, and directly viewingfluorescence emanating from the fluorescing targeting construct bound toor taken up by the tumor tissue so as to determine the location and/orsurface area of the tumor tissue in the in vivo body part. Of course, ifthe tests determine that the tumor cells are concurrently taking up morethan one tumor-avid compound in substantial proportion (e.g., bothestrogen and progesterone), the more than one tumor avid compound sodetermined can be used as the tumor-avid ligand moieties in thetargeting constructs in the invention diagnostic method.

Methods for obtaining test tumor cells for prescreening to determine thetype(s) of tumor-avid compounds that are currently being taken up (e.g.,by specific receptors expressed by the tumor cells) are well known inthe art. For example, such techniques as fine needle aspirates,scrapings, excisional biopsies, and the like, can in many instances beutilized to obtain test tumor cells relatively non-invasively.

In vitro tests useful for determining the tumor-avid compounds that arebeing taken up by test tumor cells are numerous and also well known inthe art. Such in vitro tests generally involve either sequentially orsimultaneously contacting the test cells with a plurality of differenttumor-avid compounds. For example, the test cells can be contacted witha panel or library of detectably labeled hormones and/or other knowntumor-avid compounds to determine which of the detectably labeledcompounds bind to and/or are taken up by the test cells.

In the practice of the present invention, the UV-sensitive fluorescentmoiety is linked to the tumor-avid compound used as the ligand moiety inthe targeting construct by any method presently known in the art forattaching two moieties, so long as the attachment of the linker moietyto the ligand moiety does not substantially impede binding of thetargeting construct to the target tissue and/or uptake by the tumorcells, for example, to a receptor on a cell. Those of skill in the artwill know how to select a ligand/linker pair that meets thisrequirement. For example, with regard to octreotide, it has been shownthat coupling of a linker to Tyr3 or Phe1 of octreotide does not preventthe internalization of octreotide after binding to the somatostatinreceptor (L. J. Hofland et al., Proc. Assoc. Am. Physicians 111:63-9,1999). It is also known that 1-amino-cyclobutane-1-carboxylic acid canbe tagged at the 3 carbon of the ring.

The length of the optional linker moiety is chosen to optimize thekinetics and specificity of ligand binding, including any conformationalchanges induced by binding of the ligand moiety to a target, such as anantigen or receptor. The linker moiety should be long enough andflexible enough to allow the ligand moiety and the target to freelyinteract and not so short as to cause steric hindrance between theproteinaceous ligand moiety and the target.

In one embodiment, the linker moiety is a heterobifunctional cleavablecross-linker, such as N-succinimidyl(4-iodoacetyl)-aminobenzoate;sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate;4-succinmidyl-oxycarbonyl-α-(2-pyridyldithio) toluene;sulfosuccinimidyl-6-[α-methyl-α-(pyridyldithiol)-toluamido] hexanoate;N-succinimidyl-3-(-2-pyridyldithio)-proprionate;succinimidyl-6-[3(-(-2-pyridyldithio)-proprionamido] hexanoate;sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido] hexanoate;3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine, and the like.Further bifunctional linking compounds are disclosed in U.S. Pat. Nos.5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877, each of whichis incorporated herein by reference in its entirety.

These chemical linkers can be attached to purified ligands usingnumerous protocols known in the art, such as those described in PierceChemicals “Solutions, Cross-linking of Proteins: Basic Concepts andStrategies,” Seminar #12, Rockford, Ill.

In another embodiment presently preferred, the linker moiety is apeptide having from about 2 to about 60 amino acid residues, for examplefrom about 5 to about 40, or from about 10 to about 30 amino acidresidues. This alternative is particularly advantageous when the ligandmoiety is proteinaceous. For example, the linker moiety can be aflexible spacer amino acid sequence, such as those known in single-chainantibody research. Examples of such known linker moieties include GGGGS(SEQ ID NO:1), (GGGGS)_(n) (SEQ. ID NO:2), GKSSGSGSESKS (SEQ ID NO:3),GSTSGSGKSSEGKG (SEQ. ID NO:4), GSTSGSGKSSEGSGSTKG (SEQ ID NO:5),GSTSGSGKSSEGKG (SEQ ID NO:6), GSTSGSGKPGSGEGSTKG (SEQ ID NO:7),EGKSSGSGSESKEF (SEQ ID NO:8), SRSSG (SEQ. ID NO:9), SGSSC (SEQ IDNO:10), and the like. A Diphtheria toxin trypsin sensitive linker havingthe sequence AMGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO:11) is also useful.Alternatively, the peptide linker moiety can be VM or AM, or have thestructure described by the formula: AM(G_(2 to 4)S)_(x)QAM wherein Q isselected from any amino acid and X is an integer from 1 to 11 (SEQ IDNO:12). Additional linking moieties are described, for example, inHuston et al., PNAS 85:5879-5883, 1988; Whitlow, M., et al., ProteinEngineering 6:989-995, 1993; Newton et al., Biochemistry 35:545-553,1996; A. J. Cumber et al., Bioconj. Chem. 3:397-401, 1992; Ladumer etal., J. Mol. Biol. 273:330-337, 1997; and U.S. Pat. No. 4,894,443, thelatter of which is incorporated herein by reference in its entirety.

The targeting constructs and supplemental targeting constructs used inpractice of the invention method can be administered by any route knownto those of skill in the art, such as topically, intraarticularly,intracistemally, intraocularly, intraventricularly, intrathecally,intravenously, intramuscularly, intraperitoneally, intradermally,intratracheally, intracavitarily, and the like, as well as by anycombination of any two or more thereof.

The most suitable route for administration will vary depending upon thedisease state to be treated, or the location of the suspected conditionor tumor to be diagnosed. For example, for treatment of inflammatoryconditions and various tumors, local administration, includingadministration by injection directly into the body part to be irradiatedby UV light (e.g., intracavitarily) provides the advantage that thetargeting construct (e.g., fluorescently tagged antibodies) can beadministered in a high concentration without risk of the complicationsthat may accompany systemic administration thereof.

The targeting construct is administered in a “diagnostically effectiveamount.” An effective amount is the quantity of a targeting constructnecessary to aid in direct visualization of any target tissue located inthe body part under investigation in a subject. A “subject” as the termis used herein is contemplated to include any mammal, such as adomesticated pet, farm animal, or zoo animal, but preferably is a human.Amounts effective for diagnostic use will, of course, depend on the sizeand location of the body part to be investigated, the affinity of thetargeting construct for the target tissue, the type of target tissue, aswell as the route of administration. Local administration of thetargeting construct will typically require a smaller dosage than anymode of systemic administration, although the local concentration of thetargeting construct may, in some cases, be higher following localadministration than can be achieved with safety upon systemicadministration.

Since individual subjects may present a wide variation in severity ofsymptoms and each targeting construct has its unique diagnosticcharacteristics, including, affinity of the targeting construct for thetarget, rate of clearance of the targeting construct by bodilyprocesses, the properties of the fluorophore contained therein, and thelike, the skilled practitioner will weigh the factors and vary thedosages accordingly.

The invention composition can also be formulated as a sterile injectablesuspension according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1-4, butanediol. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed, including synthetic mono- or diglycerides, fatty acids(including oleic acid), naturally occurring vegetable oils like sesameoil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fattyvehicles like ethyl oleate, or the like. Buffers, preservatives,antioxidants, and the like, can be incorporated as required, or,alternatively, can comprise the formulation.

The invention fluorescing targeting constructs can be produced by wellknown techniques. For example, well known techniques of proteinsynthesis can be used to obtain proteinaceous components of thetargeting construct if the amino acid sequence of the component isknown, or the sequence can first be determined by well known methods, ifnecessary. Some of the ligand genes are now commercially available. Anadvantage of obtaining commercially available genes is that they havegenerally been optimized for expression in E. coli. A polynucleotideencoding a protein, peptide or polynuleotide of interest, can beproduced using DNA synthesis technology. Methods for obtaining the DNAencoding an unavailable gene and expressing a gene product therefrom arewell known and will not be described here in detail.

A fluorescent targeting construct comprising a proteinaceous ligandmoiety, a proteinaceous linker moiety, and a proteinaceous fluorophorecan also be produced as a fusion protein using well known techniqueswherein a host cell is transfected with an expression vector containingexpression control sequences operably linked to a nucleic acid sequencecoding for the expression of the fusion protein (Molecular Cloning ALaboratory Manual, Sambrook et al., eds., 2nd Ed., Cold Spring HarborLaboratory, N.Y., 1989).

“Peptide” and/or “polypeptide” means a polymer in which the monomers areamino acid residues which are joined together through amide bonds,alternatively referred to as a polypeptide. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. Additionally, unnaturalamino acids such as beta-alanine, phenylglycine, and homoarginine aremeant to be included. Commonly encountered amino acids that are notgene-encoded can also be used in the present invention, althoughpreferred amino acids are those that are encodable. For a generalreview, see, for example, Spatola, A. F., in Chemistry and Biochemistryof Amino Acids, Peptides and Proteins, B. Weinstein, ed., Marcel Dekker,New York, p. 267,1983.

It will be apparent to those skilled in the art that various changes maybe made in the invention without departing from the spirit and scopethereof, and therefore, the invention encompasses embodiments inaddition to those specifically disclosed in the specification, but onlyas indicated in the appended claims.

12 1 5 PRT Artificial Sequence a peptide linker moiety 1 Gly Gly Gly GlySer 1 5 2 5 PRT Artificial Sequence a peptide linker moiety 2 Gly GlyGly Gly Ser 1 5 3 12 PRT Artificial Sequence a peptide linker moiety 3Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser 1 5 10 4 14 PRTArtificial Sequence a peptide linker moiety 4 Gly Ser Thr Ser Gly SerGly Lys Ser Ser Glu Gly Lys Gly 1 5 10 5 18 PRT Artificial Sequence apeptide linker moiety 5 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu GlySer Gly Ser Thr 1 5 10 15 Lys Gly 6 14 PRT Artificial Sequence a peptidelinker moiety 6 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly1 5 10 7 18 PRT Artificial Sequence a peptide linker moiety 7 Gly SerThr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 LysGly 8 14 PRT Artificial Sequence a peptide linker moiety 8 Glu Gly LysSer Ser Gly Ser Gly Ser Glu Ser Lys Glu Phe 1 5 10 9 5 PRT ArtificialSequence a peptide linker moiety 9 Ser Arg Ser Ser Gly 1 5 10 5 PRTArtificial Sequence a peptide linker moiety 10 Ser Gly Ser Ser Cys 1 511 28 PRT Artificial Sequence a diphtheria toxin trypsin sensitivelinker 11 Ala Met Gly Arg Ser Gly Gly Gly Cys Ala Gly Asn Arg Val GlySer 1 5 10 15 Ser Leu Ser Cys Gly Gly Leu Asn Leu Gln Ala Met 20 25 12 7PRT Artificial Sequence a peptide linker moiety having a structureselected from any amino acid and having an integer from 1 to 11 for GS12 Ala Met Gly Ser Gln Ala Met 1 5

What is claimed is:
 1. A method for in vivo identification of tumortissue associated with a disease state in a subject in need thereof,said method comprising: administering to the subject having directlyviewable tumor tissue a diagnostically effective amount of at least onebiologically compatible fluorescing targeting construct comprising atumor-avid moiety so as to allow the fluorescing targeting construct tobind to and/or be taken up selectively in vivo by the tumor tissue,irradiating an in vivo body part of the subject containing the tumortissue with UV light having at least one wavelength in the excitationspectrum of the targeting construct, and directly viewing fluorescenceemanating from the fluorescing targeting construct bound to or taken upby the tumor tissue so as to determine the location and/or surface areaof the tumor tissue in the body part.
 2. The method according to claim 1wherein the tumor-avid moiety is a hormone, a hormone receptorbinding-peptide, deoxyglucose, somatostatin, a somatostatinreceptor-binding peptide, or a combination of any two or more thereof.3. The method according to claim 1 wherein the targeting constructfurther comprises a biologically compatible UV-sensitive fluorescingmoiety.
 4. The method according to claim 2 wherein the tumor-avid moietyis somatostatin or a somatostatin receptor-binding peptide.
 5. Themethod according to claim 4 wherein the tumor tissue is anneuroendocrine or endocrine tumor.
 6. The method according to claim 4wherein the tumor tissue is melanoma, insulinoma, pancreatic tumor,small cell and non-small cell lung cancer, lymphoma, or ovarian,pituitary, pancreas, or adrenal cancer, brain tumor, colorectal cancer,cutaneous melanoma, epithelial cancer, lung carcinoma, testicular germcell tumor, or breast cancer.
 7. The method according to claim 2 whereinthe somatostatin receptor-binding peptide is octreotide, lanreotide,P587 or P829.
 8. The method according to claim 2 wherein the targetingligand is deoxyglucose.
 9. The method according to claim 8 wherein thedisease state is brain tumor, colorectal cancer, lymphoma, cutaneousmelanoma, epithelial tumors, lung carcinoma, testicular germ cell tumor,or breast cancer.
 10. The method according to claim 1 wherein thetumor-avid moiety is 1-amino-cyclobutane-1-carboxylic acid ormethionine.
 11. The method according to claim 1 wherein the in vivo bodypart is located in a body opening of the subject and wherein theirradiating of the body part and the viewing of fluorescence involves anendoscopic device to direct the UV light to the body part, and toreceive fluorescence from the fluorescing targeting construct.
 12. Themethod according to claim 1 wherein the method further comprisessurgically excising at least a part of the tumor tissue while viewingthe fluorescent image.
 13. The method according to claim 1 wherein thesurface area determined is based on the intensity of the fluorescence.14. The method according to claim 1 wherein the UV light issubstantially monochromatic and the wavelength is matched to apredominant excitation wavelength of the fluorescent targetingconstruct.
 15. The method according to claim 1 wherein the at least onewavelength of UV light is in the range from about 4 nm to about 400 nm.16. The method according to claim 11 wherein the opening is a naturalbody cavity.
 17. The method according to claim 11 wherein the opening ismade surgically.
 18. The method according to claim 11 wherein theendoscopic device is an apparatus comprising: a UV light source forgenerating a light having at least one wavelength of UV light within theexcitation spectrum of the fluorescing targeting construct; at least onetransmitting light guide adapted at the proximal end for receiving lightfrom the UV light source and transmitting the light to the distal endthereof so as to irradiate a field of vision with the UV light; at leastone receiving light guide for receiving fluorescence from the field ofvision at the distal end thereof and transmitting the fluorescence tothe proximal end thereof; and an eyepiece adapted to receive thefluorescence from the proximal end of the receiving light guide and toprovide a visual image of the fluorescence for direct viewing by anoperator.
 19. The method according to claim 11 wherein a source of theUV light is located outside of the body of the subject.
 20. The methodaccording to claim 11 wherein the endoscopic device is selected from thegroup consisting of a laparascope, a bronchoscope, a thoracoscope, anendoscope, a colonscope, a cystoscope, a culposcope, an angioscope, andan arthroscope.
 21. The method according to claim 1 wherein the viewingis for monitoring the course of the disease state.
 22. The methodaccording to claim 1 wherein the viewing identifies the tumor tissue forsurgical intervention.
 23. The method according to claim 1 wherein thesubject is located in an operating room and the conditions thatsubstantially eliminate the extraneous light comprise substantiallydarkening lights other than the UV light in the operating room.
 24. Themethod according to claim 1 wherein the method further comprisesremoving at least a portion of the tissue associated with the diseasestate.
 25. The method according to claim 24 wherein the removing issurgical removal through an opening in the body of the subject.
 26. Themethod according to claim 24 wherein the removing is by a deviceselected from the group consisting of a laparascope, a bronchoscope, athoracoscope, an endoscope, a colonscope, a cystoscope, a culposcope, anangioscope, and an arthroscope.
 27. The method according to claim 1further comprising administering to the subject at least onesupplemental fluorescing targeting construct that binds to the targetingconstruct to enhance the fluorescence.
 28. The method according to claim27 wherein the at least one supplemental fluorescing targeting constructcomprises a monoclonal antibody, or biologically active fragmentthereof.
 29. The method according to claim 28 further comprisingadministering to the subject at least one additional fluorescingantibody that binds to the fluorescent targeting construct to enhancethe fluorescence.
 30. The method according to claim 28 wherein thefragment is selected from the group consisting of Fab, Fab′, (Fab′)₂,Fv, and single chain antibody fragments.
 31. The method according toclaim 1 further comprising administering to the subject a supplementalfluorescing targeting construct that binds specifically to normal tissuein the in vivo body part, wherein fluorescence from the supplementalfluorescing targeting construct in response to the UV light is adifferent color than that from the targeting construct, and wherein thedifferent color distinguishes the tumor tissue from the normal tissue inthe body part.
 32. The method according to claim 3 wherein thefluorescing targeting construct further comprises a linker moiety forattaching the ligand moiety to the fluorescing moiety.
 33. The methodaccording to claim 32 wherein the linker moiety covalently attaches thefluorescing moiety to the fluorescing moiety.
 34. The method accordingto claim 32 wherein the linker moiety is a heterobifunctionalcrosslinker.
 35. The method according to claim 34 wherein theheterobifunctional cross-linker is selected from the group consisting ofN-succinimidyl(4-iodoacetyl)-aminobenzoate;sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate;4-succinimidyl-oxycarbonyl-α-(2-pyridyldithio)toluene;sulfosuccinimidyl-6-[α-methyl-α-(pyridyldithiol)-toluamido]hexanoate;N-succinimidyl-3-(-2-pyridyldithio)-proprionate;succinimidyl-6-[3(-(-2-pyridyldithio)-proprionamido]hexanoate;sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate;3-(2-pyridyldithio)-propionyl hydrazide, Elhman's reagent, anddichlorotriazinic acid.
 36. The method according to claim 32 wherein thelinker moiety is a peptide having from about 2 to about 60 amino acidresidues.
 37. The method according to claim 36 wherein the peptidecontains amino acid residues having a sequence selected from the groupconsisting of SEQ ID NOS: 1-10.
 38. The method according to claim 3wherein the UV-sensitive fluorescing moiety is selected from the groupconsisting of rhodamine, fluorescein, and tetracycline.
 39. The methodof claim 1 wherein the administering is by a method selected from thegroup consisting of topically, intraarticularly, intracisternally,intraocularly, intraventricularly, intrathecally, intravenously,intramuscularly, intravascularly, intercavitarily, intraperitoneally,intradermally, and by a combination of any two or more thereof.
 40. Themethod according to claim 1 wherein the administering is by localinjection.
 41. The method according to claim 1 wherein the administeringis systemic.
 42. A method for utilizing a diagnostic procedure duringsurgery in a subject in need thereof, said method comprising:administering to the subject having directly viewable tumor tissue adiagnostically effective amount of a biologically compatible fluorescingtargeting construct comprising a tumor-avid ligand moiety so as to allowthe fluorescing targeting construct to specifically bind to and/or betaken up by the tumor tissue in the subject, irradiating an in vivo bodypart of the subject containing the tumor tissue with UV light having atleast one wavelength in the excitation spectrum of the targetingconstruct under conditions that substantially eliminate extraneous lightto the body part, directly viewing fluorescence from the specificallybound and/or taken up targeting construct so as to determine thelocation and/or surface area of the tumor tissue in the body part, andremoving at least a portion of the tumor tissue.
 43. The methodaccording to claim 42 wherein the viewing of the fluorescence and theremoving of the tumor tissue are performed substantiallycontemporaneously.
 44. The method according to claim 42 wherein thetargeting construct further comprises a biologically compatibleUV-sensitive fluorescing moiety linked to the tumor-avid moiety.
 45. Themethod according to claim 42 wherein the tumor-avid moiety is a hormone,deoxyglucose, somatostatin, a somatostatin receptor-binding peptide, ora combination of any two or more thereof.
 46. A method for in vivoidentification of tumor cells associated with a disease state in subjectin need thereof, said method comprising: contacting samples of the tumorcells obtained from the subject in vitro with a plurality of detectablylabeled tumor-avid compounds, determining which of the tumor-avidcompounds bind to and/or are taken up by the sample cells, administeringto the subject a diagnostically effective amount of one or morebiologically compatible fluorescing targeting constructs, eachcomprising as ligand moiety at least one of the tumor-avid compoundsdetermined to bind to and/or be taken up by the sample cells so as toallow the fluorescing targeting construct to bind and/or by taken upselectively in vivo by directly viewable tumor tissue, irrading an invivo body part of the subject containing the tumor tissue with UV lighthaving at least one wavelength in the excitation spectrum of thetargeting construct under conditions that substantially eliminateextraneous light to the in vivo body part, and directly viewingfluorescence emanating form the fluorescing targeting construct bound toand/or taken up by the tumor tissue so as to determine the locationand/or surface area of the tumor tissue in the directly viewable in vivobody part.